摘要
编码完整大肠杆菌不耐热肠毒素 (LT)的基因被引入pET11c形成pET11 LT ,该质粒在E .coliBL2 1(DE3)中得到较高效率的表达 ,约 4 6mg L。用D(+) Immobilizedgalactose柱可以在很宽的pH范围 (pH7 3～ 10 4 )内用多种方法对LT进行纯化且保持其结构完整。溶于TEAN(pH7 3)或碳酸盐缓冲液 (pH10 4 )的LT ,冻干后保存于 4℃ ,可长久保持其完整结构 ,此为保存LT的较好策略。与GM1结合实验、CHO细胞及Patent mouse毒性检测实验证明纯化的LT具有生物学活性。
Heat labile enterotoxin(LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E.coli BL21(DE3). By growing this strain on modified M9 CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+) Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN(pH 7 3) containing 0 3mol/L galactose and carbonate buffer(pH 10 4) containing 0 3mol/L galactose. After dried by freeze and placed in 4℃, LT dissolved in TEAN(pH 7 3) and carbonate buffer(pH 10 4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biolo gical character.
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第5期532-537,共6页
Chinese Journal of Biotechnology
关键词
大肠杆菌
不耐热肠毒素
表达
纯化
保存
heat labile enterotoxin, expression, purification, storage
